Mahtab Z. Siddiqui

Conformation of a T-cell stimulating peptide of interleukin-1β protein: circular dichroism studies

A T-cell stimulating peptide Val-Gln-Gly-Glu-Glu-Ser-Asn-Asp-Lys-OH, the 163-171 fragment epitope of interleukin-1 beta (IL-1 beta), has been synthesized in solution phase and purified by reverse-phase high-performance liquid chromatography (RP-HPLC). The backbone conformation of the synthetic fragment, investigated in aqueous solution by circular dichroism (CD) spectroscopy, is qualitatively a mixture of beta-turns and random coil. Quantification of the CD spectra revealed the presence of a 9% beta-turn fraction in water at pH 7.0, suggesting the occurrence of the conformation for the epitope fragment in aqueous solution necessary for T-cell stimulation and antigenicity. Concomitant changes in CD spectra were observed with increases in the trifluoroethanol (TFE) concentration in water, and the beta-turn fraction in peptide increased to 28% at a concentration of 90% TFE. This helicogenic solvent, as well as other solvents such as methanol, acetonitrile and dioxane (all favouring an ordered structure in peptides), failed to induce any alpha-helical conformation in the IL-1 beta (163-171) fragment, and CD spectra were attributed to only beta-turn ordered structure. This beta-turn structure has also been found to be a theoretically preferred conformation using Chou-Fasman proclivity data and is in accordance with the presence of an all-beta-globular conformation for its parent molecule IL-1 beta. Thus, the beta-turn conformation is probably involved in retention of T-cell stimulation activity in this synthetic epitope.

Solution conformation of tuftsin.

Conformation of chemically synthesized tetrapeptide tuftsin, an important immunomodulator, with the sequence Thr-Lys-Pro-Arg-OH has been studied in aqueous solution as well as in a number of organic solvents viz. trifluoroethanol (TFE), methanol and dioxane by circular dichroism (CD) spectroscopy which reveals preferential occurrence of mixture of random coil and beta-turns, that has already been predicted as bioactive conformation of this peptide.

DEHYDROPHENYLALANINE ANALOGUES : CONFORMATIONAL CHARACTERIZATION

Few dehydrophenylalanine (deltaPhe) analogues (X-deltaPhe-Phe-Gly-X1, X = Ac-; Boc-; Z-; X1 = OMe; OH; ONH2) of virus replication inhibiting peptide (Z-D-Phe-Phe-Gly) were synthesized, and their solution conformations were investigated by 1H NMR, UV and circular dichroism (CD) spectroscopy. Homogeneity of these analogues was also assessed by reverse phase-high performance liquid chromatography (RP-HPLC) using water-acetonitrile gradients.

Detection and Partial Purification of Salmonella serovar Typhimurium Cytotoxic Protein Affecting Seed Germination

Abstract Siddiqui, M.Z., Singh, B.R., Chandra, M., Agarwal, Ravi Kant, Agarwal, R.K. and Srivastava, S.K. 2007. Detection and partial purification of Salmonella serovar Typhimurium cytotoxic protein affecting seed germination. J. Appl. Anim. Res., 33: 77–79. Cells of a virulent strain of Salmonella Typhimurium (E-2391), a highly ubiquitous and zoonotic serovar, were sonicated and the cell-free supernatant was precipitated using increasing concentration of ammonium sulphate (30–80%). Inhibition of seed germination by various precipitated fractions revealed that the activity was present in the fraction precipitated at 70% saturation of ammonium sulphate. Out of the two peaks obtained on Seralose 6B chromatography, the second peak was found to possess germination-inhibition activity at a level as low as 0.1 mg/ml of this protein. SDS-PAGE revealed the presence of fewer proteins in the second peak but no specific protein responsible for the inhibition could be deduced. The study indicated that the in vitro seed germination inhibition technique can be utilized for testing of Salmonella isolates for the cytotoxic properties instead of resorting to expensive in vivo methods involving use of animals.

Intra-Species Mice Hybridomas Against a Recombinant Protein of Mycobacterium avium paratuberculosis

Abstract Singh, M., Siddiqui, M.Z. and Singh, R.P. 2002. Intra-species mice hybridomas against a recombinant protein of Mycobacterium avium paratuberculosis. J. Appl. Anim. Res., 22: 137–144. Intra-species mice hybridomas were produced by fusing the 35-kDa recombinant protein of Mycobacterium avium paratuberculosis primed splenocytes of Swiss white mice with BALB/c derived myeloma cells. Presence of peritoneal macrophages derived from Swiss white mice as feeder layer did not make much difference in the production of intra-species mice hybridomas. The total hybridoma production efficiency was 71% when peritoneal macrophages were used as feeder layer, whereas it was 59% when no feeder layer was used. Five stable positive hybridomas were subcloned to produce monoclonals. Out of 5 monoclonal antibodies (MAbs) obtained, 4 MAbs (1AG2, 2BD2, 2BG2, 1DE8) were of IgGl sub-class, whereas the fifth MAb (1AG8) was IgG2b. The Western blot analyses of these MAbs demonstrated the specificity of the antibodies to the recombinant protein through the presence of strongly reacting bands on immunoblots.

Isolation and Partial Characterization of Buffalo Plasma Fibronectin (Fn)

Abstract Jagati, R., Paliwal, S., Siddiqui, M.Z., Kumar, S., Sharma, A. K. and Kumar, A. 1995. Isolation and partial characterization of buffalo plasma fibronectin (Fn). J. Appl. Anim. Res., 8: 97–104. Fibronectin (Fn) a large extracellular adhesive glycoprotein was isolated from buffalo plasma using gelatin-sepharose affinity chromatography and characterized by analytical SDS-PAGE, GPC-HPLC and amino acid analysis. Analytical GPC-HPLC revealed homogeneity of the isolate with a molecular weight of 460 kD and SDS-PAGE analysis of Fn in reduced form depicted presence of two 230 kD polypeptide chains linked by disulphide bonds. Microchemical analysis of the Fn hydrolysate using 6N HCl gave the amino acid composition similar to that of other species except a higher content of arginine (R) i.e. 8.2% as compared to 5.3% in other species. Staphylococcus aureus cells showed preferential binding to Fn coated surface.